characterization of multiple c2 domain and transmembrane … · multiple c2 domain and...

Characterization ofMultiple C2Domain and TransmembraneRegion Proteins in Arabidopsis1[OPEN]

Lu Liu, Chunying Li, Zhe Liang, and Hao Yu2

Department of Biological Sciences and Temasek Life Sciences Laboratory, National University of Singapore,117543, Singapore

ORCID IDs: 0000-0003-4353-1795 (L.L.); 0000-0002-0006-419X (C.L.); 0000-0001-6872-6463 (Z.L.); 0000-0002-9778-8855 (H.Y.).

Multiple C2 domain and transmembrane region proteins (MCTPs) are evolutionarily conserved in eukaryotic organisms andmay function as signaling molecules that mediate trafficking of other regulators. Although there are a large number of MCTPsfound in the plant lineage, biological information on most plant MCTPs remains unknown. Here, we report systematiccharacterization of 16 members in the entire Arabidopsis (Arabidopsis thaliana) MCTP family. Using GUS and GFP reporterassays, we reveal their distinct or overlapping patterns of gene expression and protein localization in developing Arabidopsisplants and Nicotiana benthamiana leaf epidermal cells. We further analyze in vivo effects of three C2 domains on the regulatoryrole of MCTP1 (FTIP1) in flowering time control in Arabidopsis, demonstrating that these C2 domains may be cooperative tomediate FTIP1 function during the floral transition. Through examining all available T-DNA insertional mutants of ArabidopsisMCTPs, we further reveal that mctp6-1 significantly enhances the late-flowering phenotype of ftip1-1 possibly through affectingFLOWERING LOCUS T in different manners, exemplifying that different MCTPs additively regulate a specific plantdevelopmental process. Taken together, our results suggest functional divergence or redundancy of MCTP members inArabidopsis and provide a community resource for further understanding various MCTP functions in plant development.

Division or differentiation of plant cells is highlydetermined by the positional and developmental in-formation perceived by these cells. Cell-to-cell com-munication plays an important role in this process andis crucial to mediate signals from diverse pathways.Transduction of extracellular signals by receptor-likekinases or direct transfer of transcription factors be-tween cells is part of this sort of communication re-quired for modulating plant growth and developmentin response to environmental and internal signals (VanNorman et al., 2011; Wu and Gallagher, 2012), which isusually regulated by both intercellular and intracellulartrafficking pathways.

Plants have evolved an elaborate endomembranesystem for proper compartmentation of signalingmolecules that facilitate trafficking of proteins or othermacromolecules among endomembrane compartments

(Contento and Bassham, 2012). Recent findings havesuggested that a group of highly conservedmultiple C2domain and transmembrane region proteins (MCTPs),each of which contains three to four C2 domains at theN terminus and one to four transmembrane regions atthe C terminus, could function as important signalingmolecules that mediate trafficking of other regulators inplant cells (Shin et al., 2005; Fulton et al., 2009; Liu et al.,2012, 2013; Song et al., 2017). The C2 domain is one ofthe most prevalent eukaryotic lipid binding domainsand could serve as a docking module that targets pro-teins to specific intracellular membrane by formingphospholipid complexes (Nalefski and Falke, 1996). Inmembrane trafficking proteins, different C2 domainsalways bear different conserved sequences, implyingthat multiple C2 domains in these proteins may func-tion cooperatively rather than additively for a specificcellular function (Cho and Stahelin, 2006).

Sequence analyses have revealed that while MCTPsare evolutionarily conserved (Nalefski and Falke, 1996;Shin et al., 2005; Lek et al., 2012; Liu et al., 2013), there isa significantly increased number of MCTP repertoire inthe plant lineage compared to the number of counter-parts in animals. This implies more diverse and specificfunctions of MCTPs in regulating cellular processes inplants. There are a total of 16MCTPs in the Arabidopsis(Arabidopsis thaliana) genome, among which QUIRKY(QKY) and FT-INTERACTING PROTEIN1 (FTIP1) havebeen suggested to affect macromolecular trafficking(Fulton et al., 2009; Liu et al., 2012). QKY interacts with aLeu-rich repeat receptor-like kinase STRUBBELIG (SUB),thus affecting intercellular communication that contributes

1 This work was supported by the Singapore National ResearchFoundation Investigatorship Programme (NRF-NRFI2016-02) and byintramural research support from the National University ofSingapore and Temasek Life Sciences Laboratory.

2 Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Hao Yu ([email protected]).

L.L. and H.Y. conceived and designed the experiments; L.L., C.L.,and Z.L. performed the experiments; L.L., C.L., Z.L., and H.Y. ana-lyzed the data; L.L. and H.Y. wrote the article.

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Plant Physiology�, March 2018, Vol. 176, pp. 2119–2132, www.plantphysiol.org � 2018 American Society of Plant Biologists. All Rights Reserved. 2119

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http://orcid.org/0000-0001-6872-6463

http://orcid.org/0000-0001-6872-6463

http://orcid.org/0000-0002-9778-8855

http://orcid.org/0000-0002-9778-8855

http://orcid.org/0000-0002-9778-8855

http://orcid.org/0000-0003-4353-1795

http://orcid.org/0000-0002-0006-419X

http://orcid.org/0000-0001-6872-6463

http://orcid.org/0000-0002-9778-8855

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to the relevant plant organogenesis mediated by SUB(Fulton et al., 2009; Trehin et al., 2013; Vaddepalli et al.,2014). FTIP1 is associated with endoplasmic reticulum(ER) membrane and mediates intercellular trafficking ofthe florigen protein FLOWERING LOCUS T (FT) specifi-cally from companion cells to sieve elements, thus con-trolling flowering time in Arabidopsis (Liu et al., 2012).The closest ortholog of FTIP1 in rice (Oryza sativa),OsFTIP1, plays a similar role in mediating rice floweringtime under long days through affecting trafficking of a ricecounterpart of FT, RICE FLOWERING LOCUS T1, fromcompanion cells to sieve elements (Song et al., 2017).Interestingly, both QKY and FTIP1 have been found to bespecifically localized in plasmodesmata that are importantconduits for exchange of molecules among plant cells (Liuet al., 2012; Vaddepalli et al., 2014), suggesting that theseMCTPs might serve as essential regulators for mediatingkey intercellular signaling through plasmodesmata.

To systematically investigate MCTPs in Arabidopsis,in this studywe used both GUS andGFP reporter assaysto explore gene and protein expression of 16members inthe entire Arabidopsis MCTP family, respectively. Weexamined dynamic tissue- and developmental-specificGUS expression driven by 59 upstream sequences ofMCTP members and revealed their distinct or over-lapping expression patterns in developing Arabidopsisplants. We also investigated the subcellular localizationof MCTP members by transiently expressing GFP-MCTPs in Nicotiana benthamiana leaf epidermal cells. Tounderstand the function of various C2 domains inMCTPs, we further analyzed in vivo effects of three C2domains on the regulatory role of FTIP1 in floweringtime control in Arabidopsis. In addition, through ex-amining all available T-DNA insertional mutants ofArabidopsisMCTPs,we showed thatmctp6-1 significantlyenhanced the late-flowering phenotype of ftip1-1 possiblythrough affecting FT, exemplifying thatMCTPs additivelyregulate a specific plant developmental process. Takentogether, our results demonstrate functional divergence orredundancy of MCTP members in Arabidopsis and es-tablish a community resource for further understandingMCTP function in macromolecular trafficking in plants.

RESULTS

Identification of MCTP Family Proteins in Arabidopsis

As QKY and FTIP1 were the first two MCTP proteinsidentified in Arabidopsis (Fulton et al., 2009; Liu et al.,2012), we used the FTIP1 protein sequence as a query tosearch for putative MCTPs using BLAST program inThe Arabidopsis Information Resource and identified atotal of 16MCTPmembers, designatedMCTP1-16, eachof which contains three to four N-terminal C2 domainsand one to four C-terminal transmembrane regions(Fig. 1; Supplemental Fig. S1).

We classified these 16 Arabidopsis MCTPs into fiveclades (Fig. 1) through phylogenetic analysis based onmultiple sequence alignment. These 16 MCTPs share

relatively conserved amino acids in the three C2 do-mains and the transmembrane regions (SupplementalFig. S2), while the overall amino acid identities amongthese family members ranged from 33% to 93%(Supplemental Fig. S3). Notably, theN-terminal regionsof these MCTPs contain highly variable amino acidsequences with variable lengths (Supplemental Fig. S2),indicating that the N-terminal region may contribute tothe functional divergence among MCTPs.

In silico chromosome mapping showed that the16 MCTP genes were dispersed throughout the Arabi-dopsis genome and located in all the chromosomesexcept chromosome II (Supplemental Fig. S4). Inter-estingly, the closest homologs, such as MCTP3 andMCTP4, are located separately in different chromo-somes. This scattered distribution of closest homologsis possibly due to the extensive reshuffling and diver-gent evolution following massive duplications.

Phylogenetic Analysis of MCTP Homologs in Plants

To identify MCTP homologs in plant genomes, weperformed BLASTP or TBLASTN search in protein andgenome databases using FTIP1 as a query sequence andfound MCTP homologs in all land plant genomes thathave been sequenced. The evolutionary relationshipamong MCTPs in different species was analyzed indetail using neighbor-joining and maximum likelihoodmethods. We included the angiosperm eudicot Arabi-dopsis, the monocot rice, the gymnosperm Norwayspruce (Picea abies), lycophyte (Selaginella moellendorffii),and moss (Physcomitrella patens) in phylogenetic anal-ysis (Fig. 2). It is noteworthy that MCTPs exist in bothlycophytes, which are the oldest living lineage of vas-cular plants, and mosses, which are the early nonvas-cular land plants. These MCTPs are present in aseparate clade representing its early formation inseedless plants. The number of MCTPs in seed plants isgreatly expanded and classified into five groups (I–V)based on strong bootstrap values (Fig. 2). In contrast,there are a limited number of MCTPs in animals, suchas human and mouse (Supplemental Fig. S5). A largenumber of MCTP repertoires in the plant lineage implythat as immovable organisms, plants may evolve togenerate more functionally specialized MCTPs to me-diate cellular activities in response to changing devel-opmental or environmental stimuli.

Analysis of pMCTP:GUS Activity in ArabidopsisVegetative Tissues

Among all MCTP genes in Arabidopsis, only theexpression patterns of MCTP1 (FTIP1) and MCTP15(QKY) have already been reported (Liu et al., 2012;Trehin et al., 2013; Vaddepalli et al., 2014). To under-stand temporal and spatial expression patterns of allArabidopsis MCTP genes, we generated pMCTP:GUSreporter lines, in which the GUS gene was driven by 1.5

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to 3 kb of the 59 genomic region upstream of each MCTPcoding sequence. At least 15 independent transgenic lineswere created for each construct. Since most of the trans-genic lines for each construct showed consistent GUSstainingpatterns,we selected one representative line eachfor further monitoring the detailed expression patterns invarious tissues at different developmental stages. Ingeneral, none of the pMCTP:GUS reporter lines exhibitedconstitutive expression patterns, while the 59 upstreamsequences of MCTPs in the same clade often conferreddistinct GUS expression patterns in various tissues, sug-gesting diverse functions of MCTPs in various develop-mental processes in the life cycle of Arabidopsis.We first examined pMCTP:GUS reporter lines at the

vegetative phase by staining of 11-d-old whole seedlings(Fig. 3; Supplemental Fig. S6A) and roots of 5-d-oldseedlings (Fig. 4; Supplemental Fig. S6, B and C). Alllines except for MCTP11 displayed GUS staining tovarious extents in either shoots and/or roots. In the ae-rial part, MCTP1 (FTIP1), MCTP2, MCTP3, MCTP6,MCTP15 (QKY), and MCTP16 lines all displayed GUSactivity in vascular tissues (Fig. 3, A–C, F, O, and P).Their staining patterns were grouped into three differenttypes. GUS expression ofMCTP3,MCTP6, andMCTP16lines was early detected in whole protruding youngleaves, but mainly found in vascular tissues of adultleaves at later stages (Fig. 3, C, F, and P). GUS staining ofMCTP1 (FTIP1) andMCTP15 (QKY) lines was relativelyuniform in vascular tissues throughout cotyledons androsette leaves (Fig. 3, A and O), whereas staining of theMCTP2 line was relatively weak in secondary and ter-tiary veins compared to primary veins (Fig. 3B).MCTP3,MCTP4, MCTP5, MCTP6, and MCTP16 lines exhibitedGUS staining in the shoot apex region (Fig. 3, C–F and P),while MCTP9, MCTP10, MCTP13, and MCTP14 linesdisplayedGUS activity in incipient leaf primordia (Fig. 3,I, J, M, and N). The MCTP7 line exhibited GUS stainingspecifically in the hydathode region (Fig. 3G).All pMCTP:GUS reporter lines except for those con-

taining MCTP11 and MCTP13 upstream sequencesshowed various GUS staining patterns in roots (Fig. 4).

Compared to other lines, therewas relatively specificGUSactivity detected in root vascular tissues of MCTP1(FTIP1),MCTP2,MCTP12,MCTP15 (QKY), andMCTP16lines, among which only GUS staining of the MCTP1(FTIP1) line did not extend to the meristematic zone (Fig.4, A, B, L, O, and P). MCTP4, MCTP5, MCTP10, andMCTP14 lines exhibited strong GUS activity throughoutthe meristematic zone (Fig. 4, D, E, J, and N), whereasGUS activity of theMCTP7 line was only restricted in thebasal meristem (Fig. 4G). MCTP6 and MCTP8 lines werethe only ones that displayed GUS activity in the root cap(Fig. 4F) and root hair (Fig. 4H), respectively.

Analysis of pMCTP:GUS Activity in ArabidopsisReproductive Tissues

Except for MCTP8 and MCTP13, the upstream se-quences of the other MCTPs were able to drive GUSexpression with diverse patterns in inflorescences bear-ing flowers at various stages (Figs. 5 and 6;Supplemental Fig. S6, D and E). There were consistentGUS staining patterns detected in flowers at differentstages in many MCTP lines, such as MCTP1 (FTIP1),MCTP3, MCTP6, and MCTP16 (Fig. 5, A, C, F, and P),whereas other lines showed changes in GUS staining indeveloping flowers. For example, GUS staining of theMCTP9 line was only detected in flowers at late stages(Figs. 5I and 6I). In contrast, GUS activity of theMCTP14line was only observed in stamens of young flowers, butdisappeared in old flowers (Figs. 5N and 6N). Interest-ingly, both MCTP7 and MCTP11 lines displayed dy-namic changes in GUS expression in various floralorgans at different stages. GUS staining of the MCTP7line was detected in gynoecia of young flowers, but laterconfined to the distal end of filaments of old flowers(Figs. 5G and 6G). GUS activity of the MCTP11 linegradually decreased in pollen grains, but increased inovules of developing flowers (Figs. 5K and 6K).

While most of the pMCTP:GUS reporter linesexhibited GUS staining patterns that were consistent

Figure 1. Classification of MCTP family proteinsin Arabidopsis. Five groups of MCTP proteins aredefined based on phylogenetic analysis of Arabi-dopsisMCTPs shown on the left. The phylogenetictree was generated with MEGA6 using theneighbor-joining algorithm. Numbers on the ma-jor branches indicate bootstrap values (.50%) in1,000 replicates. For each MCTP, the ArabidopsisGenome Initiative (AGI) gene number, othername, amino acid number, molecular weight, pIof the predicted protein, and schematic diagramof major motifs are indicated in the table on theright. The prediction of protein motifs is basedon SMART (http://smart.embl-heidelberg.de). C2domains and transmembrane (TM) regions arelabeled as green and blue boxes, respectively. aa,Amino acids.

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with the gene expression data available in ArabidopsiseFP browser (Winter et al., 2007), inconsistent patternswere found for MCTP14. MCTP14 was ubiquitouslyexpressed in Arabidopsis based on the microarray data ineFP browser, while the pMCTP14:GUS line displayedspecific staining mainly in incipient leaf primordia at thevegetative stage (Fig. 3N) and in stamens of youngflowers(Fig. 5N). It is possible that the 59 upstream sequence in-cluded in the pMCTP14:GUS constructmaynot contain allessential cis-elements that are required for conferring en-dogenous MCTP14 expression in Arabidopsis.

Subcellular Localization of the MCTPs in Arabidopsis

To determine the subcellular localization of16 MCTPs, we transiently expressed their full-lengthopen reading frames fused with the GFP reporter(GFP-MCTPs) in N. benthamiana leaf epidermal cells(Fig. 7) and observed generally four different types ofsubcellular localization patterns in tobacco cells. Thefirst group of MCTPs, including MCTP1 (FTIP1),MCTP5, MCTP10, MCTP11, MCTP12, and MCTP16,were associated with the ER network (Fig. 7, A, E, J–L,and P), which is substantiated by colocalization of

Figure 2. Phylogenetic relationshipsofMCTPhomologs indifferentplant species. Proteins fromdifferent species are indicatedbya two-letterprefix (AT, Arabidopsis thaliana; Os,Oryza sativa; Pa, Picea abies; Sm, Selaginella moellendorffii; Pp, Physcomitrella patens). All availablegene names are also indicated. For each node, the level of statistical support by the neighbor-joiningmethod using the Jones-Thornton-Taylorwith discrete Gamma distribution (+G) model and the maximum likelihood method inferred using the LG+G model is marked by a filledcircle if both values are above 80% bootstrap support or an open circle if both values are higher than 50% bootstrap support.


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representative MCTPs in this group with an ERmarker, RFP-HDEL (Supplemental Fig. S7A; Nelsonet al., 2007). The second group of MCTPs comprisingMCTP3, MCTP4, MCTP6, MCTP9, MCTP13, andMCTP15 (QKY) was detected in both the cytosol andplasmamembrane (Fig. 7, C, D, F, I, M, and O). MCTPsin the third group, including MCTP7, MCTP8, andMCTP14, were only localized in small structureswithin cells, possibly intracellular vesicles (Fig. 7, G,H, and N), while MCTP2 in the last group was onlylocalized in the plasma membrane (Fig. 7B). To furtherunderstand the concrete subcellular localization ofMCTPs in the second and third groups, we coex-pressed 35S:GFP-MCTPswith the fluorescence-taggedorganelle markers for Golgi (35S:Man49-CFP; Nelsonet al., 2007) and endosomes (35S:RFP-RabF2b; Jaillaiset al., 2006). We observed that MCTP3, MCTP4,MCTP6, and MCTP7 partially resided in the endo-somal compartments (Supplemental Fig. S7B), whileMCTP14 and MCTP15 were partially localized in the

Golgi (Supplemental Fig. S7C). Distinct subcellularlocalization patterns of the MCTPs in tobacco cellsindicate that different MCTPs may exert distinctfunctions in plant cells.

Characterization of C2 Domains in MCTP1 (FTIP1)

Although multiple C2 domains have been suggestedto either additively or individually act to mediate pro-tein functions (Damer and Creutz, 1994; Cho and Sta-helin, 2006; Marty et al., 2013), it is unclear howmultiple C2 domains play roles in mediating MCTPfunctions in plants. To this end, we examined the role ofeach C2 domain in MCTP1 (FTIP1), which affectsflowering time through mediating FT trafficking fromcompanion cells to sieve elements (Liu et al., 2012).

As the N-terminal region of FTIP1 containing threeC2 domains interacts with FT (Liu et al., 2012), wetested the interaction of each C2 domain in the

Figure 3. GUS staining of 11-d-old pMCTP:GUS lines grown under long days. Representative GUS staining of pMCTP1:GUS (A),pMCTP2:GUS (B), pMCTP3:GUS (C), pMCTP4:GUS (D), pMCTP5:GUS (E), pMCTP6:GUS (F), pMCTP7:GUS (G), pMCTP8:GUS(H), pMCTP9:GUS (I), pMCTP10:GUS (J), pMCTP11:GUS (K), pMCTP12:GUS (L), pMCTP13:GUS (M), pMCTP14:GUS (N),pMCTP15:GUS (O), and pMCTP16:GUS (P). The inset in (G) shows a higher magnification of leaves with GUS staining in hy-dathodes (arrowheads). Bar = 2 mm.








N-terminal region with FT by yeast two-hybrid assaysand found that only the third C2 domain interactedwith FT (Fig. 8, A and B), suggesting that this C2 do-main is specifically required for FTIP1 interaction withFT. Since both FTIP1 and FT are colocalized to ER inplant cells (Liu et al., 2012), we further transientlyexpressed various truncated versions of FTIP1-GFP ei-ther containing individual C2 domains and the trans-membrane region or with deletion of specific domain(s)in N. benthamiana leaf epidermal cells (Fig. 9A) toidentify key domains required for FTIP1 subcellular

localization (Fig. 9B). The GFP fusion proteins con-taining individual C2 domains were localized in boththe plasmamembrane and nucleus (Fig. 9B, panels 1–3),while the fusion protein bearing the C-terminal trans-membrane region was obviously associated with theER network (Fig. 9B, panel 4), demonstrating an im-portant role of the transmembrane region in conferringFTIP1 localization into ER. Furthermore, among theFTIP1-GFP truncated proteins with deletion of individualdomains, only deletion of the secondC2domain or part ofthe transmembrane region compromised FTIP1-GFP

Figure 4. GUS staining of the meristematic (left panel) and elongation zones (right panel) of pMCTP:GUS roots 5 d after ger-mination grown on Murashige and Skoog plates under long days. Representative GUS staining of pMCTP1:GUS (A), pMCTP2:GUS (B), pMCTP3:GUS (C), pMCTP4:GUS (D), pMCTP5:GUS (E), pMCTP6:GUS (F), pMCTP7:GUS (G), pMCTP8:GUS (H),pMCTP9:GUS (I), pMCTP10:GUS (J), pMCTP11:GUS (K), pMCTP12:GUS (L), pMCTP13:GUS (M), pMCTP14:GUS (N),pMCTP15:GUS (O), and pMCTP16:GUS (P). Bar = 100 mm.


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association with the ER network (Fig. 9B, panels 5–8).These results suggest that both the C-terminal trans-membrane region and the secondC2 domain of FTIP1 areessential for subcellular localization of FTIP1 into ER, al-though the second C2 domain itself is not sufficient forconferring this localization.FTIP1 functions to mediate FT trafficking in the

phloem so that the late-flowering phenotype of ftip1-1 canbe rescued by the expression of FTIP1 coding sequencedriven by the promoter of SUC TRANSPORTER2(SUC2), which is active specifically in the phloemcompanion cells (Fig. 8C; Imlau et al., 1999; Liu et al.,2012). To examine the effect of each C2 domain onFTIP1 role in promoting flowering, we transformedftip1-1 with various pSUC2:FTIP1 truncated con-structs containing deletion of each C2 domain. Inter-estingly, all ftip1-1 lines transformed with the truncated

versions of FTIP1 exhibited comparable flowering timeto ftip1-1 (Fig. 8C), demonstrating that all the three C2domains are essential for FTIP1 function in promotingflowering.

FTIP1 and MCTP6 Function Additively to PromoteFlowering under Long Days

To understand the biological functions of ArabidopsisMCTPs during the floral transition, we isolated theavailable T-DNA insertional lines for 15MCTPs from theArabidopsis Biological Resource Center (SupplementalFig. S8) and observed their growth phenotypes underlong days. In addition tomctp1-1 ( ftip1-1; SALK_013179)and mctp15 (qky-14; SALK_061045) that exhibited thelate-flowering phenotype as previously reported (Liuet al., 2012; Trehin et al., 2013),mctp6-1 (SALK_145386)

Figure 5. GUS staining of inflorescence apices of pMCTP:GUS lines grown under long days. Representative GUS staining ofpMCTP1:GUS (A), pMCTP2:GUS (B), pMCTP3:GUS (C), pMCTP4:GUS (D), pMCTP5:GUS (E), pMCTP6:GUS (F), pMCTP7:GUS(G), pMCTP8:GUS (H), pMCTP9:GUS (I), pMCTP10:GUS (J), pMCTP11:GUS (K), pMCTP12:GUS (L), pMCTP13:GUS (M),pMCTP14:GUS (N), pMCTP15:GUS (O), and pMCTP16:GUS (P). Bar = 3 mm.







exhibited a similar flowering defect under long days(Fig. 10, A–D; Supplemental Fig. S8).

The full-length MCTP6 transcript was undetectablein mctp6-1 (Fig. 10B). Single mctp6-1 mutants floweredslightly later than wild-type plants, but significantlyenhanced the late-flowering phenotype of ftip1-1 onlyunder long days, whereas all of these single and doublemutants did not exhibit a flowering defect under shortdays (Fig. 10, C and D), suggesting that FTIP1 andMCTP6 function additively to promote flowering spe-cifically under long days. Consistently, we found thatMCTP6 expression exhibited a diurnal oscillation underlong dayswith peaks at night (Fig. 10E). Daylength shiftexperiments showed that like FT, expression ofMCTP6was substantially increased when shifted from shortdays to long days (Fig. 10F), demonstrating thatMCTP6expression is promoted by the photoperiod pathway.Furthermore, during the floral transition underlong days, which occurred around 9 to 13 d after seed

germination under our growth conditions, GUS stainingof pMCTP6:GUS lines was consistently strong in vascularand mesophyll tissue of young leaves, but weak in oldercotyledons and leaves (Fig. 3F; Supplemental Fig. S9).

To test whether MCTP6 and FTIP1 may share similarfunction to affect FT, we performed yeast two-hybridassays and revealed the interaction of the truncatedMCTP6 containing only the four C2 domains at the Nterminus with FT and TWIN SISTER OF FT (TSF), butnot with TERMINAL FLOWER1 (TFL1), all of whichbelong to the phosphatidylethanolamine-binding pro-tein family (Fig. 10, G and H; Kobayashi et al., 1999;Wigge et al., 2005). In addition, we found that likeFTIP1-GFP (Liu et al., 2012), GFP-MCTP6 was colo-calizedwith callose deposition indicated by aniline bluestaining, marking localization of GFP-MCTP6 in plas-modesmata (Fig. 10I). These observations imply thatFTIP1 andMCTP6 could play additive roles in affectingFT during the floral transition.

Figure 6. GUS staining of open flowers ofpMCTP:GUS lines grown under long days.Representative GUS staining of pMCTP1:GUS (A), pMCTP2:GUS (B), pMCTP3:GUS(C), pMCTP4:GUS (D), pMCTP5:GUS (E),pMCTP6:GUS (F), pMCTP7:GUS (G),pMCTP8:GUS (H), pMCTP9:GUS (I),pMCTP10:GUS (J), pMCTP11:GUS (K),pMCTP12:GUS (L), pMCTP13:GUS (M),pMCTP14:GUS (N), pMCTP15:GUS (O),and pMCTP16:GUS (P). Bar = 2 mm.


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DISCUSSION

Proteins containing multiple C2 domains and trans-membrane region(s) are evolutionarily conserved anddivided into four subgroups, including synaptotagmins,ferlins, tricalbins, andMCTPs, based on the number of C2domains and the location of transmembrane region(s)(Lek et al., 2012). Although there are only a limitednumber of MCTPs, each containing three to four C2 do-mains at the N terminus and one to four transmembrane

regions at the C terminus, in unicellular ancestors andanimals, the number of MCTPs is significantly increasedin higher plants (Fig. 2; Supplemental Fig. S5; Nalefskiand Falke, 1996; Shin et al., 2005; Lek et al., 2012; Liu et al.,2013). This may cater to the requirement of more func-tionally specific MCTPs to mediate plant response tochanging internal or environmental stimuli. Although afew plant MCTPs have been shown to be involved inmembrane trafficking and fusion processes (Fulton et al.,

Figure 7. Subcellular localization of GFP-MCTPs in N. benthamiana leaf epidermal cells. Representative GFP fluorescenceimages of MCTP1 (FTIP1)-GFP (A), GFP-MCTP2 (B), GFP-MCTP3 (C), GFP-MCTP4 (D), GFP-MCTP5 (E), GFP-MCTP6 (F), GFP-MCTP7 (G), GFP-MCTP8 (H), GFP-MCTP9 (I), GFP-MCTP10 (J), GFP-MCTP11 (K), GFP-MCTP12 (L), GFP-MCTP13 (M), GFP-MCTP14 (N), GFP-MCTP15 (O), and GFP-MCTP16 (P). Bar = 30 mm.






2009; Liu et al., 2012, 2013; Vaddepalli et al., 2014; Songet al., 2017), biological information on most of plantMCTPs remains unknown.

In this study, we have systematically characterized16 MCTPs in Arabidopsis, which are grouped into fiveclades based on the phylogenetic analysis (Fig. 1). Ex-amination of the GUS reporter lines driven by the 59upstream sequences of these MCTPs has revealed thatnone of the pMCTP:GUS reporter lines exhibit exactlythe same GUS staining patterns at both vegetative andreproductive tissues. Notably, 59 upstream sequencesof MCTPs even in the same clades often drive GUSexpression with distinct patterns in various tissues. Forexample, while MCTP1 (FTIP1) and MCTP2 lines gen-erally exhibit GUS staining in vascular tissues, theirdistribution patterns in vegetative or reproductive tis-sues are different. In the aerial part of vegetative seed-lings, GUS activity is uniformly detected in vasculartissues of cotyledons and rosette leaves of the MCTP1(FTIP1) line (Fig. 3A), whereas GUS staining of theMCTP2 line is obvious in primary veins (Fig. 3B). Inroots, although GUS activity is detectable in vasculartissues of both MCTP1 (FTIP1) and MCTP2 lines, theMCTP1 (FTIP1) line displays stronger GUS staining inthe elongation zone than theMCTP2 line (Fig. 4, A andB). However, in the root meristematic zone, GUSstaining completely disappears in the MCTP1 (FTIP1)line but is obvious in the MCTP2 line (Fig. 4, A and B).These different expression patterns indicate that evenMCTPs sharing high sequence similarity might be dif-ferentially regulated to play distinct roles in varioustissues. This is further substantiated by distinct sub-cellular localization patterns of GFP-MCTPs revealed inN. benthamiana leaf epidermal cells (Fig. 7).

Despite differential GUS expression patternsdriven by the 59 upstream sequences of differentMCTPs, some pMCTP:GUS reporter lines display

overlapping GUS activity in specific tissues. For ex-ample, MCTP1 (FTIP1), MCTP2, MCTP3, MCTP6,MCTP15 (QKY), and MCTP16 lines all exhibit GUSactivity in vascular tissues of vegetative seedlings,albeit to different extents (Fig. 3, A–C, F, O, and P),implying that some MCTPs may act redundantly oradditively in specific tissues during Arabidopsis de-velopment. Similarly, comparable subcellular locali-zation exhibited by some MCTPs also indicates thatsome of them may participate redundantly or addi-tively in similar cellular processes (Fig. 7). Thus,distinct or overlapping patterns of gene expressionand subcellular localization revealed by our GUS andGFP reporter assays could be further investigated toexplore functional divergence or redundancy ofMCTP members in Arabidopsis.

The presence of multiple C2 domains at the N termi-nus is a signature feature of MCTPs, but their concretefunctions in MCTPs remain unknown. Characterizationof multiple C2 domains in MCTP1 (FTIP1) in this studyhas revealed specific roles of each C2 domain in FTIP1(Figs. 8 and 9). Deletion of each C2 domain in FTIP1 al-most completely compromises FTIP1 role in promotingflowering, suggesting that all the three C2 domains arerequired for FTIP1 function in the control of floweringtime in Arabidopsis. Notably, the third C2 domain isspecifically required for FTIP1 interaction with FT, whilethe second C2 domain, together with the C-terminaltransmembrane region, is essential for MCTP1 (FTIP1)localization into the ER network. Since C2 domain hasbeen suggested to act as a docking module targetingproteins to specific intracellular membrane (Nalefski andFalke, 1996), our findings suggest that multiple C2 do-mains inMCTPsmay be cooperative to play an importantrole inmediatingMCTP interactionwith target protein(s),their subcellular localization, and subsequent biologicaleffects.

Figure 8. Function of C2 domains of FTIP1 in thecontrol of flowering time. A, Yeast two-hybridassay of interaction between FT and each C2domain of FTIP1. Transformed yeast cells weregrown on SD-Trp/-Leu medium (left panel) andSD-His/-Trp/-Leu medium supplemented with10 mM 3-amino-1,2,4-trizaole (right panel). B,Quantification of interaction between FT andeach C2 domain of FTIP1 in yeast by b-galacto-sidase assays. C, Distribution of flowering time inT1 transgenic plants carrying the full-length ortruncated versions (without each C2 domain) ofthe FTIP1 coding sequence in ftip1-1 back-ground.


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Through examining the available T-DNA insertionallines for 15 Arabidopsis MCTPs, we have found thatmost of these lines do not display obvious defects underour growth conditions, implying possible functionalredundancy among these MCTPs. Three T-DNA inser-tional lines, including mctp1-1 (ftip1-1) (Liu et al., 2012),mctp15 (qky-14) (Trehin et al., 2013), and mctp6-1, showthe late-flowering phenotype (Fig. 10, A–D), suggestingthat FTIP1, QKY, andMCTP6 are individually requiredfor promoting flowering. Several pieces of evidenceindicate that FTIP1 andMCTP6 affect flowering time indifferent manners. First, their gene expression patternsin tissues are fundamentally different. FTIP1 is specifi-cally expressed in vascular tissues of cotyledons androsette leaves, but not in the shoot apex (Liu et al., 2012;Fig. 3A), whereasMCTP6 is expressed in the shoot apexand early detected in whole protruding young leaves(Fig. 3F). Second,MCTP6 expression shows an obviouscircadian rhythmunder long days (Fig. 10E), which is in

contrast to the nonrhythmic pattern of FTIP1 (Liu et al.,2012), indicating that they may respond differently tothe photoperiod signal. Third, the subcellular localiza-tion patterns of FTIP1 and MCTP6 are also different.FTIP1 is associated with the ER network, whereasMCTP6 is mainly in intracellular compartments and theplasma membrane (Fig. 7, A and F). These observationssuggest that FTIP1 and MCTP6 contribute to floweringtime control in different ways at both cellular and tissuelevels. Consistently, mctp6-1 significantly enhances thelate-flowering phenotype of ftip1-1 under long days(Fig. 10, C and D). Despite these differences, FTIP1 andMCTP6 do share some common characteristics, such asinteractionwith FT, localization in plasmodesmata, andfunctioning in the photoperiod pathway. Thus, it willbe interesting to further investigate their roles in thefloral transition to understand whether these twoMCTPs could play either simple additive or morecomplex cooperative roles in affecting FT.

Figure 9. Subcellular localization of truncated FTIP1 proteins in N. benthamiana leaf epidermal cells. A, Schematic diagramsdepicting 8 different truncated versions of FTIP1 proteins fused with GFP as shown in B. C2 domains and transmembrane (TM)regions are labeled as cyan and orange boxes, respectively. B, Subcellular localization of 8 different truncated versions of FTIP1-GFP fusion proteins in N. benthamiana leaf epidermal cells. GFP, GFP fluorescence; BF, bright field; Merge, merge of GFP andbright field. Bar = 10 mm.





Figure 10. The late-flowering phenotype of ftip1-1 is enhanced by mctp6-1. A, Schematic diagram shows the T-DNA insertionsite inmctp6-1 (SALK_145386). The exon and 59/39 untranslated regions are indicated by black and gray boxes, respectively. Thestart codon (ATG) and stop codon (TAA) are labeled. B, MCTP6 expression is undetectable in mctp6-1 by semiquantitative PCRusing the primers flanking the T-DNA insertion site. TUB2 expression is used as a control. C, ftip1-1 mctp6-1 flowers later thanftip1-1 or mctp6-1 under long days. D, Comparison of flowering time of mctp6-1, ftip1-1, and mctp6-1 ftip1-1 under long days(LDs) and short days (SDs). Error bars indicate SD. Single asterisks indicate a significant difference in flowering time between singlemutants and wild-type plants, while a double asterisk indicates a significant difference in flowering time between double andsingle mutants (Student’s t test, P , 0.05). E, MCTP6 expression exhibits obvious diurnal oscillation in 9-d-old wild-type plantsgrown under LDs harvested at 2-h intervals over a 24-h period. FT expression was examined as a control. Sampling time wasexpressed in hours as Zeitgeber time (ZT), which is the number of hours after the onset of illumination. The lowest expression levelof each gene is set as 1. Bars below the graph indicate the duration of day (white) and night (black). Error bars denote SD. F,MCTP6expression is induced upon daylength extension.Wild-type seedlingswere grown under SDs for 11 d before theywere transferredto LDs.MCTP6 expression in seedlings were examined by quantitative real-time PCR at 4-h intervals for 3 d comprising one SDfollowed by two LDs. FT expression was examined as a control. The lowest expression level of each gene is set as 1. Error barsdenote SD. G, Yeast two-hybrid assay of interaction between FT, TSF, or TFL1 and the N-terminal region of MCTP6 containing fourC2 domains. Transformed yeast cells were grown on SD-Trp/Leu medium (left panel) and SD-His/-Trp/-Leu medium supple-mented with 5 mM 3-amino-1,2,4-triazole (right panel). H, Quantification of interaction of FT, TSF, or TFL1 and the N-terminalregion of MCTP6 containing four C2 domains in yeast by b-galactosidase assays. I, Subcellular localization of GFP-MCTP6 and


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CONCLUSION

In conclusion, our systematic analysis of 16 genes inthe entire Arabidopsis MCTP family illustrates theircomplex gene expression and protein localization pat-terns in Arabidopsis. Through examining the roles ofmultiple C2 domains in FTIP1, we show that thesesignature domains in MCTPs may be cooperative tomediate MCTP function in plant development. In ad-dition, various MCTPs, such as FTIP1 and MCTP6,could act in different manners to affect the same targetto regulate a specific developmental process. Takentogether, our findings reveal potential functional di-vergence or redundancy of MCTP members in Arabi-dopsis and establish a community resource for furthermechanistic understanding MCTP function in plants.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Arabidopsis (Arabidopsis thaliana) plants in the Columbia (Col) backgroundwere grown under long days (16 h light/8 h dark) or short days (8 h of light/16 h of dark) at 23°C. All T-DNA insertional mutants in the Col backgroundwere obtained from the Arabidopsis Biological Resource Center (http://www.arabidopsis.org).

Sequence Analysis

A primary BLASTP or TBLASTN search was performed using the Arabi-dopsis FTIP1 as a query sequence against protein and genome databases(Phytozome database, https://phytozome.jgi.doe.gov; Spruce genomedatabase, http://congenie.org) to identify FTIP1 homologs. The sequencescontaining C2 domain and transmembrane regionswere all included for furtheranalysis. Each sequence was then searched against the protein ConservedDomain Database, SMART Database, and the Pfam Database to annotate do-main modules. The program ProtTest was used for estimating the best modelfor each alignment by default, which identified LG+I+G+F as the best fit of the112 examined evolutionary models according to Akaike Information Criterionstatistics. The neighbor-joining phylogeny was performed by MEGA version6.0 with 1,000 replicates. Since LG+I+G+F is not available in MEGA, we usedthe next best available JJT + G model and pairwise deletion. The maximumlikelihood phylogeny was performed by PhyML software with 100 replicatesand the best available LG + G model.

Plasmid Construction

pMCTP1(FTIP1):GUS and 35S:MCTP1(FTIP1)-GFP constructs were previ-ously described (Liu et al., 2012). To generate other pMCTP:GUS reporterconstructs, 1.5 to 3 kb of 59 upstream sequence of each MCTP were amplifiedand cloned into pHY107 (Liu et al., 2007). To generate other 35S:GFP-MCTPreporter constructs, the cDNA encoding each full-length MCTP or each domainwas amplified and cloned into pGreen-35S-GFP. To create various truncatedversions of the constructs, pSUC2:FTIP1 and 35S:FTIP1-GFP (Liu et al., 2012)were mutagenized using a modified QuikChange site-directed mutagenesisapproach. The primers used for plasmid construction are listed in SupplementalTable S1. Transgenic plants were generated in the Col backgroundthrough Agrobacterium tumefaciens-mediated transformation and selected byBasta on soil.

GUS Staining

GUS staining was performed as previously reported with minor modifica-tions (Yu et al., 2000). Tissues were infiltrated with staining solution (50 mM

sodium phosphate buffer, pH 7.0, 0.5 mM potassium ferrocyanide, 0.5 mM

potassium ferricyanide, and 0.5 mg/mL X-Gluc) in a vacuum chamber andsubsequently incubated with staining solution at 37°C overnight.

Transient Expression of Proteins in Tobacco LeafEpidermal Cells

The overnight A. tumefaciens cultures with expression vectors were har-vested and resuspended in the infiltration buffer (10 mM MES, pH 5.6, 10 mM

MgCl2, and 100 mM acetosyringone) with OD600 at 0.2. Infiltration solutionswere infiltrated into the abaxial surface of 3-week-old tobacco (Nicotiana ben-thamiana) leaves with syringes. The leaves were examined 2 d after infiltrationunder confocal microscope.

Yeast Two-Hybrid Assay

To construct the vectors for yeast two-hybrid assays, the coding sequencesof FT, each C2 domain of FTIP1,MCTP6 (N750), TSF, and TFL1were amplifiedand cloned into pGADT7 or pGBKT7 (Clontech). The yeast two-hybridassay was performed using the Yeastmaker Yeast Transformation System2 (Clontech).

Expression Analysis

Total RNA was extracted using the FavorPrep Plant Total RNA Mini Kit(Favorgen) and reverse transcribed using the M-MLV Reverse Transcriptase(Promega). Quantitative real-time PCR was performed on three biologicalreplicates using 7900HTFast Real-TimePCR systems (Applied Biosystems)withMaxima SYBR Green/ROX qPCR Master Mix (Fermentas). The relative ex-pression levels normalized to TUB2were calculated as previously reported (Liuet al., 2007). Primers for real-time PCR are listed in Supplemental Table S1.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data-bases under the following accession numbers: MCTP1 (FTIP1), At5g06850;MCTP2, At5g48060; MCTP3, At3g57880; MCTP4, At1g51570; MCTP5,At5g12970; MCTP6, At1g22610; MCTP7, At4g11610; MCTP8, At3g61300;MCTP9, At4g00700; MCTP10, At1g04150; MCTP11, At4g20080; MCTP12,At3g61720; MCTP13, At5g03435; MCTP14, At3g03680; MCTP15 (QKY),At1g74720; MCTP16, At5g17980; TFL1, At5g03840; TSF, At4g20370; and FT,At1g65480.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Topology prediction of transmembrane helices of16 MCTPs in Arabidopsis.

Supplemental Figure S2. Alignment of amino acid sequences of 16 MCTPsin Arabidopsis.

Supplemental Figure S3. Homology matrix tree of 16 MCTPs in Arabi-dopsis.

Supplemental Figure S4. Location of the MCTP Homologs on the Arabi-dopsis chromosomes.

Supplemental Figure S5. Phylogenetic analysis of MCTPs in animals.

Figure 10. (Continued.)free GFP in N. benthamiana leaf epidermal cells. Compared to free GFP, GFP-MCTP6 signal is colocalized with callose depo-sition staining with aniline blue, which indicates the position of plasmodesmata. GFP, GFP fluorescence; AB, aniline bluestaining; BF, bright field image; Merge, merge of GFP, AB, and BF. Bar = 5 mm.




http://www.arabidopsis.org

http://www.arabidopsis.org

https://phytozome.jgi.doe.gov

http://congenie.org










Supplemental Figure S6. GUS staining in various tissues of wild-typeplants grown under long days.

Supplemental Figure S7. Colocalization of representative GFP-MCTPswith various subcellular markers in N. benthamiana leaf epidermal cells.

Supplemental Figure S8. List of T-DNA insertion mutants of ArabidopsisMCTPs.

Supplemental Figure S9. GUS staining of developing pMCTP6:GUS seed-lings grown under long days.

Supplemental Table S1. List of primers used in this study.

ACKNOWLEDGMENTS

We thank the Arabidopsis Biological Resource Center for providing seedsand members of the Yu lab for discussion and comments on the manuscript.

Received August 14, 2017; accepted December 17, 2017; published December19, 2017.

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